PROJECT SUMMARY SLE is a disease of high morbidity for which treatment options are poor. Identifying relevant targets for therapeutic intervention is difficult owing to the molecular and clinical heterogeneity of the disease. Through a large body of work, we have demonstrated that blood-based transcriptomic profiling can be used to parse out distinct transcriptional differences in immune function in health and disease. Relevant to the current application are our studies showing that SLE can be divided into seven disease subgroups based on distinct blood transcriptional signatures. More recently, we developed a sophisticated for integrated Profiling and Analysis Pipeline (iPAP) that we used to identify meaningful epigenetic and transcriptomic differences in human populations, including SLE cohorts. Building upon this knowledgebase will help us identify potential drivers of the disease, and ultimately novel biomarkers and novel therapeutic targets for SLE. We hypothesize that SLE is comprised of a spectrum of disease subtypes that can be categorized according to mutually distinct epigenomic, isoformic and transcriptomic features. The goal of this project is to characterize the repertoire of immune cell isoforms and the epigenome profiles associated with three of the seven most frequent pediatric SLE subgroups: SLE-Plasmablast (SLE-P), characterized by a plasmablast transcriptional signature and the presence of increased circulating plasmablasts; SLE-Interferon/Myeloid cells (SLE-IM), which shows an IFN and a neutrophil signature; and SLE-Plasmablast/ Interferon/Myeloid cells (SLE-PIM) displaying the three main signatures. We will use novel experimental paradigms that incorporate cutting-edge genomic technologies and build upon our established expertise in molecular immune profiling. Our project contains two Specific Aims: 1) To analyze the alternatively spliced transcriptome of blood cells from the three SLE patient subgroups, in which we will apply both short-read (Illumina HiSeq2500) and long-read (PacBio RSII) sequencing technologies to interrogate the isoform repertoire of PBMCs and select immune cell subsets; and 2) To determine the epigenome of blood cells from the three SLE subgroups, through studies capitalizing on a robust and sensitive new epigenomic assay called ATAC-seq coupled to Methyl-seq. Healthy age- matched samples will be used as controls in all experiments and, we will also examine samples from children with active juvenile dermatomyositis and systemic juvenile arthritis (n=5 per group), to identify those isoforms and signatures unique to SLE from those more generally associated with autoimmunity. Our team includes experts in immunology, genomics, epigenetics, systems biology of disease, computational biology, data management and statistics, who bring the collective expertise necessary for generating top quality genomics data and for robust data analysis using state-of-the-art methods. We will also create new tools, i.e., Nanostring probes and monoclonal antibodies, for high throughout screening assays suited to large patient cohorts. Successful completion of our project will reveal novel biomarkers of SLE and/or novel therapeutic targets.